Cardiomyocyte hypertrophy is a common endpoint of heart aging and many cardiac diseases. It is not clear what triggers the change and the mechanism of the change. Oxidants are by-products of aerobic metabolism and increase in the heart under the pathological conditions associated with ischemia-reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that low concentrations of H2O2 can cause cardiomyocyte hypertrophy. H2O2 treated cells can be 4 times bigger in volume or 4-10 times larger in cell surface area and contain 3 times more proteins per cell. Cardiomyocyte hypertrophy induced by endocrine factors involves elevated expression of a number of genes, activation of MAP kinases and calcium mediated activation of NF-AT3. Since H2O2 has been reported to activate MAP kinases and increase cytosolic calcium, we hypothesize that H2O2 activates MAP kinases or calcium dependent NF-AT3 transcription factor to alter the expression of hypertrophic genes and to induce cardiomyocyte hypertrophy. The expression of the atrial natriuretic factor, beta-myosin heavy chain, skeletal alpha-actin, alpha-myosin heavy chain, ventricular-specific myosin light chain-2 and troponin C genes will be measured by Northern blot in H2O2 treated H9C2 cells. The activity of the stress activated protein kinase, extracellular regulated kinase and p38 MAP kinase will be determined after H2O2 treatment. The inhibitors of these kinases will be tested for their effect on induction of hypertrophic genes and cell enlargement by H2O2. In parallel, cytosolic calcium concentration and DNA binding activity of NF-AT3 will be measured after treating cells with H2O2. Calcium chelators and inhibitors of calcineurin will be tested for their ability to prevent H2O2 induced N-FAT3 activation, activation of hypertrophic genes and cell enlargement. We propose to study the mechanism of oxidant stress by novel and innovative approaches. The results will contribute to the understanding of the process of heart aging and the pathogenesis of heart failure.